Crocodilepox virus protein 157 is an independently evolved inhibitor of protein kinase R
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Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 May 2022 doi:10.20944/preprints202205.0393.v1
Crocodilepox virus protein 157 is an independently evolved inhibitor
of protein kinase R
M. Julhasur Rahman1,2, Loubna Tazi1, Sherry L. Haller3 and Stefan Rothenburg1*
1
School of Medicine, University of California Davis, Department of Medial Microbiology and
Immunology, Davis, California, United States of America
2
current address: Purification Development Department, Genentech, Inc., One DNA Way, South
San Francisco, California, United States of America
3
University of Texas Medical Branch at Galveston, Center for Biodefense and Emerging Infectious
Diseases, Galveston, Texas, United States of America
*
Address for correspondence: Stefan Rothenburg, School of Medicine, University of California
Davis, Department of Medial Microbiology and Immunology, Davis, CA 95616.
rothenburg@ucdavis.edu. ORCID iD:0000-0002-2525-8230
Keywords: poxviruses, protein kinase R, evolution, translational regulation, eIF2
1
© 2022 by the author(s). Distributed under a Creative Commons CC BY license.Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 May 2022 doi:10.20944/preprints202205.0393.v1
Abstract
Crocodilepox virus (CRV) belongs to the Poxviridae family and mainly infects hatchling and
juvenile Nile crocodiles. Most poxviruses encode inhibitors of the host antiviral protein kinase R
(PKR), which is activated by viral double-stranded (ds) RNA formed during virus replication,
resulting in the phosphorylation of eIF2a and subsequent shutdown of general mRNA translation.
Because CRV lacks orthologs of known poxviral PKR inhibitors, we experimentally characterized
one candidate (CRV157), which contains a predicted dsRNA-binding domain. Bioinformatic
analyses indicated that CRV157 evolved independently from other poxvirus PKR inhibitors.
CRV157 bound to dsRNA, co-localized with PKR in the cytosol, and inhibited PKR from various
species. To analyze whether CRV157 could inhibit PKR in the context of a poxvirus infection, we
constructed recombinant vaccinia virus strains that contain either CRV157 or a mutant CRV157
deficient in dsRNA binding in a strain that lacks PKR inhibitors. The presence of wild type
CRV157 rescued vaccinia virus replication, while the CRV157 mutant did not. The ability of
CRV157 to inhibit PKR correlated with virus replication and eIF2a phosphorylation. The
independent evolution of CRV157 demonstrates that poxvirus PKR inhibitors evolved from a
diverse set of ancestral genes in an example of convergent evolution.
2Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 May 2022 doi:10.20944/preprints202205.0393.v1
1. Introduction
The members of the Poxviridae family are large double-stranded DNA viruses, which exclusively
replicate in the host cytoplasm. The genomes of poxviruses range between 134 kb and 360 kb and
encode approximately 200 genes [1]. Poxvirus members can infect vertebrates and invertebrates,
collectively exhibiting a broad host range. Whereas some members strictly infect one or a limited
number of host species, others can infect a large number of host species. For example, variola virus
(VARV), the causative agent of smallpox disease, only productively infects humans. In contrast,
other poxviruses, including cowpox viruses (CPXV), monkeypox virus (MPXV), and vaccinia
virus (VACV) can infect a wide range of host species [2-4].
Poxvirus infections have been reported worldwide in host species from the Crocodylia order,
including caimans (Spectacled and Brazilian caiman) and crocodiles (Nile, saltwater, and
freshwater crocodiles), especially in farmed caiman and crocodiles [5-9]. So far, two poxviruses
infecting Crocodylia have been completely sequenced and assigned to the Crocodilydpoxvirus
genus of the Chordopoxvirinae subfamily: crocodilepox virus (CRV) isolated from farmed Nile
crocodiles [10] and saltwater crocodilepox virus (SwCRV) isolated from Australian saltwater
crocodiles [11]. CRV mainly infects hatchling and juvenile Nile crocodiles and causes scattered,
circular, grey to white skin lesions on the dorsal and ventral parts of the body, including the hind
feet, mouth cavity, eyes, nostrils, head and tail regions [8,12,13]. The lesions on the eyes and head
can impair vision and lead to skin deformities. These skin lesions can lead to further opportunistic
infections by Dermatophilus-like bacteria or fungi. Although, in general the mortality rate is low
in Nile crocodiles, the morbidity rate and economic impact in farmed crocodiles is high [12,14].
Morphologically, CRV and SwCRV virions are similar to virions of orthopoxviruses, the most
widely studied genus of Chordopoxvirinae. The virions are brick-shaped and contain a dumbbell-
shaped central core, which is flanked by lateral bodies [6,15,16]. The genome of CRV is 190,054bp
long and, unlike orthopoxviruses, contains a high GC content (62%). A total of 173 protein coding
genes have been predicated in the CRV genome, which contains two short (1754 bp) inverted
terminal repeats (ITRs). An interesting feature of CRV is that its genome lacks recognizable
homologs of most host-immune evasion genes found in other chordopoxviruses suggesting that it
evolved unique genes for manipulating the antiviral host response. CRV encodes 62 predicted
proteins that don’t show apparent sequence homology with non-Crocodylidpoxvirus proteins.
Forty-eight of these protein encoding genes are located in the terminal regions [10].
The ability of poxviruses to enter host cells is independent of species-specific cellular receptors,
which allows poxviruses to be able to enter into many different cell types [2,17]. The successful
replication of poxviruses depends on their ability to subvert host immune responses [2,3]. During
virus infections, host cells induce type I interferons (IFNs) and other antiviral host pathways,
leading to the establishment of an antiviral state [18]. Poxviruses accommodate a variety of
different immunomodulatory genes, which interfere with host antiviral proteins to allow virus
replication [19]. One of the IFN-induced host antiviral proteins is protein kinase R (PKR), which
is targeted by many viruses, including poxviruses [20-24]. PKR is present at intermediate levels in
unstimulated cells as an inactive monomer. Upon detection and binding of double-stranded (ds)
RNA produced during virus replication and transcription, PKR undergoes conformational changes,
which lead to PKR activation via homodimerization and autophosphorylation. Activated PKR
phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), which leads
to the suppression of general mRNA translation and in turn restricts virus replication [25-32]. In
addition, PKR can also inhibit virus replication by inducing proinflammatory responses and cell
death in infected cells [33-36]. Most mammalian poxviruses inhibit PKR by encoding orthologs of
3Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 May 2022 doi:10.20944/preprints202205.0393.v1
two vaccinia virus PKR inhibitors: E3 and K3 [37,38]. E3 inhibits PKR activation by binding to
dsRNA and inhibiting PKR homodimerization [22,39-41]. K3 is a pseudosubstrate inhibitor of PKR,
which restricts eIF2a and PKR interactions [21,23,42].
CRV lacks homologs of known PKR inhibitors [10]. It is therefore unknown how it inhibits the
PKR pathway. To investigate this, we performed a BLAST database searches and identified a
putative viral PKR inhibitor from open reading frame (ORF) 157 of the CRV genome, CRV157.
CRV157 was previously characterized in silico as a putative dsRNA binding protein [43]. CRV157
contains a putative dsRNA binding domain (dsRBD) and an uncharacterized C-terminal domain
that does not show homology to other known proteins. In this study, we investigated the hypothesis
that CRV157, with its putative dsRNA binding domain, might act as a PKR antagonist. The results
shown here indicate that CRV157 can bind dsRNA, inhibit PKRs from different species, and that
its dsRNA binding ability is essential for PKR inhibition. Furthermore, CRV157 was able to rescue
replication of a recombinant VACV that lacks both PKR inhibitors E3L and K3L. These results
indicate that CRV is an independently evolved PKR inhibitor.
2. Materials and Methods
2.1 Cell lines
European rabbit RK13 (kindly provided by Dr. Bernard Moss), HeLa (ATCC#CCL-22), HeLa-
PKRkd (knockdown) (kindly provided by Dr. Charles Samuel [35], and RK13+E3+K3 (RK13 cells
stably express VACV E3L and K3L) [44] cell lines were maintained in Dulbecco’s Modified
Essential Medium (DMEM, Life Technologies) supplemented with 5% fetal bovine serum (FBS,
Fischer Scientific) and 25µg/ml gentamycin (Quality Biologicals). RK13+E3+K3 cells were
additionally supplemented with 500µg/ml geneticin (Life Technologies) and 300µg/ml zeocin
(Life Technologies). All cells were incubated at 37° C, 5% CO2.
2.2 Plasmids
ORFs of all PKRs from the indicated species and viral genes were cloned under the control of a
SV40 promoter into the pSG5 vector (Stratagene) for transient expression. Construction of
knockdown resistant human PKR, mouse PKR, European rabbit PKR, and Chinese hamster PKR
was previously reported [45,46]. Chicken PKR (identical to XM_015283611.2) gene was cloned
from a 14-day old chicken embryo (Gallus gallus). Armenian hamster PKR and pig PKR were
cloned from AHL-1 cells (ATCC-CCL-195) and swine PK15 cells (ATCC-CCL-33), respectively.
CRV157 (GeneID: 4363410) was obtained from gBlocks®Gene fragments (IDT) and cloned into
the TOPO-TA vector (Invitrogen). The CRV157 ORF was then subcloned into the pSG5 vector
with SacI and XhoI restriction sites. CRV157-K48A/K49A, CRV157 delC, and CRV157 delN
plasmids were generated by site-directed mutagenesis or deletion mutation PCR using pfu high-
fidelity DNA polymerase (Invitrogen). For site-directed mutagenesis, PCR was performed using
pSG5-CRV157 as a template with the primers containing lysine to alanine mutations (AAA to
GCA and AAG to GCG) from nucleotide position 142 to 146). For the C-terminal deletion,
nucleotide position 1 to 198 and N-terminal deletion, nucleotide position 199 to 384 were PCR
amplified and cloned into pSG5 vector. To make flag epitope-tags at the C-termini of VACV E3L,
CRV157, CRV157-K48A/K49A, CRV157 del C, and CRV157 del N plasmids, these ORFs were
cloned into a pSG5 vector containing two in-frame flag tag sequences. CRV157-mCherry and
4Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 May 2022 doi:10.20944/preprints202205.0393.v1
chicken PKR-EGFP fusion genes were constructed by fusion PCR, and constructs were cloned
into the pSG5 vector. All plasmid constructs were sequenced to confirm the correct sequences.
2.3 Transfection and luciferase-based reporter (LBR) assays
Luciferase assays were performed following the protocol described previously [45]. Briefly, HeLa-
PKRkd cells (5X104 cells/ well) were seeded in 24 well plates a day before the transfection. For
each transfection reaction mix, 0.05µg of firefly luciferase (Promega) and indicated PKR (0.2µg)
plasmids were co-transfected with 0.8µg of either E3L, CRV157, CRV157-K48A/K49A, CRV157
del C, or CRV157 del N plasmids using GenJet (Signagen) with transfection reagent to DNA ratios
of 2:1. For titration experiments, indicated concentrations of CRV157 or CRV157 del C plasmids
were co-transfected. Each transfection was done in triplicate, and plasmid DNA concentration for
each transfection mix was kept constant by adding an appropriate amount of pSG5 vector plasmid
where necessary. After 48 h, whole cell lysates were extracted using mammalian lysis buffer
(Goldbio) followed by the addition of luciferin (Promega). Luciferase activity was determined in
a Glowmax luminometer (Promega). Each experiment was conducted at least three times, and
representative experiments are shown.
2.4 Viruses and infection assays
Vaccinia Virus Copenhagen strain, VC-2, was used as a parental strain to make all the recombinant
viruses used in this study. VC-2 and its derivative vP872 (∆K3L) [21] were kindly provided by Dr.
Bertram Jacobs. The construction of the vP872 derivative VC-R4 (∆E3L, ∆K3L, in which E3L
was replaced by EGFP) was described previously [47]. Recombinant viruses including VC-R4-
VACV E3L, VC-R4-CRV157 and VC-R4-CRV157-K48A/K49A were generated into the VC-R4
backbone by scarless integration of the indicated genes into the E3L locus of VC-R4 as described
[47]. All the recombinant viruses were plaque purified three times. PCR amplification and
sequencing of recombinant virus integration sites were done to confirm successful integration and
correct sequence of CRV157, VC-R4-CRV157-K48A/K49A, or E3L.
Replication kinetics of VC-R4 recombinants were tested in the RK13 cell line. Briefly, RK13 cells
(6 X 105 cells) were plated in 6-well plates a day before the infection. RK13 cells were infected at
a MOI of 0.01 with VC-R4, or VC-R4 derived recombinant viruses. Virus samples were sonicated
before the infection (2 times for 15s) and diluted in 2.5% FBS supplemented DMEM media. Each
infection at each different time point was done in duplicate. Growth medium from each well was
aspirated to add virus inocula to respective wells and then incubated at 37˚C for 1 hour. After 1-
hour incubation period, virus inocula were removed, and each well was washed twice with
phosphate buffered saline (PBS), and fresh DMEM media supplemented with 5% FBS was added.
Cells were then scraped and collected along with the media at 0, 12, 24, 48, 72, and 96 hpi.
Collected viruses were then subjected to three rounds of freeze/thaw cycles followed by sonication
(2 times for 30s, 50% amplitude) in a cup sonicator (Qsonica Q500). Titration of collected viruses
was done in RK13+E3+K3 cells by measuring plaque forming units per ml (pfu/ml).
2.5 Poly I:C Pull-down assays
Pull-down assays were performed following the protocol previously described [48]. Sepharose
beads were prepared by dissolving one gram of lyophilized CNBr-activated sepharose powder
beads (17-0430-01; GE Healthcare Life Sciences) in 40 mL of 1mM HCl followed by mixing 10
times by end-to-end rotation. The beads were then pelleted by centrifuging at 1000 X g for 5
minutes; the supernatant was then removed without disturbing the beads. The addition of 1mM
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HCl and removal of supernatant after centrifugation was repeated 4 times. After the final wash,
HCl was removed, and the beads were ready for Poly I:C conjugation. A stock solution of poly I:C
was prepared in coupling buffer (0.1 M NaHCO3, 0.5M NaCl, pH 8.3) at 10mg/mL concentration.
One gram of prepared sepharose beads was mixed with 2mL of poly I:C stock solution in a total
volume of 4mL in coupling buffer and mixed by end over end rotation at RT for 2 hours.
Unconjugated poly I:C was removed by washing the beads 5 times with equal volumes of coupling
buffer and centrifugation at 1000 X g for 5 minutes between washes and removal of the
supernatant. Beads were then incubated with 0.1M Tris HCl, pH 8 for 2 hours at RT to block the
unconjugated active sites on the beads followed by 3 cycles of washing of conjugated beads with
alternating low-pH (0.1Msodium acetate, 0.5M NaCl, pH 4.0) and high-pH (0.1MTris-HCl,
0.5MNaCl, pH 8.0) buffer (5 volumes of bead volume of each pH buffer were used). After the
final wash, beads were stored in Co-IP lysis buffer (20mMTris-HCl, pH 8, 137 mM NaCl, 10%
glycerol, 1% Triton-X, 2 mM EDTA) before use as a 50% bead slurry (equal volume of buffer
added to the beads).
HeLa-PKRkd cells were transfected at 70%-80% confluency in 6-well plates with 3µg of plasmids
pSG5-flag, pSG5-E3L-flag, pSG5-CRV157-flag or pSG5-CRV157-K48A/K49A-flag. After 48-
hours, the media was removed and washed with PBS buffer followed by cell lysis with 300 µl of
lysis buffer (20mM Tris-HCl, pH 8, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA,
protease inhibitor) on ice. For the pre-pull-down assay, 30µl of lysed samples were separated, and
the rest of the samples were centrifuged at 5400 X g for 10 minutes to remove cellular debris. Pull-
down of poly I:C was done by adding 50µl of poly I:C bead slurry to each cell lysate sample
followed by end over end rotation at RT for 2 hours and subsequent centrifugation at 5400 X g for
2 minutes and removal of the supernatant. The samples were then washed 3 times with 200 µl of
lysis buffer, and after the final wash, proteins were released from the poly I:C beads, samples were
resuspended in 120 µl of Laemmli buffer (6X SDS, β-mercaptoethanol) and denatured at 95˚C for
10 minutes. The samples were then centrifuged at 5400 X g for 2 min, and supernatants were
collected and subjected to Western blotting.
2.6 Western blot
Protein lysates from HeLa-PKRkd cells transfected with the indicated plasmid constructs (3µg)
were collected 48 hours post transfection in 1% sodium dodecyl-sulfate (SDS) in PBS (VWR).
Protein lysates from RK13 cells infected with either VC-R4 (∆E3L∆K3L), VC-2, VC-R4-VACV
E3L, VC-R4-CRV157, or VCR2-CRV157-K48A/K49A at MOI= 3 were collected at 6 hours post
infection (hpi) in 1% SDS. All protein lysates were sonicated (twice for 5s at 50% amplitude) to
shear genomic DNA, separated by 12% SDS- polyacrylamide gel electrophoresis and blotted onto
Hybond-PVDF (GE Healthcare) membranes using a methanol-based wet transfer apparatus
(BioRad). Membranes were blocked for 1 hr at room temperature with either SuperBlock blocking
buffer (ThermoFisher) when detecting flag and ß-actin, 5% (wt/vol), BSA when detecting
phosphorylated eIF2a, or 5% (wt/vol) nonfat milk when detecting total eIF2a in TBST buffer (20
mM Tris, 150 mM NaCl, and 0.1% Tween-20 pH 7.4). The blots were then incubated overnight at
4˚C with primary antibody either mouse anti-D (1:1000, FLAG, abm), rabbit eIF2a-P (1:1000,
Santa Cruz Biotechnology, sc-101670), rabbit total eIF2a (1:1000, Santa Cruz Biotechnology, sc-
11386) or mouse anti-b-actin (1:1000, Sigma-Aldrich) diluted in TBST buffer. The membranes
were then washed three times for 5 minutes each in 1X TBST buffer followed by incubation with
horseradish peroxidase conjugated secondary antibodies (donkey anti-rabbit-HRP or donkey anti-
mouse-HRP, Life Technologies) in blocking buffer for 1 h at room temperature. The membranes
6Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 May 2022 doi:10.20944/preprints202205.0393.v1
were then washed 3 times 10 minutes each in TBST buffer. After washing the membranes, proteins
bound by the secondary antibody were detected with Proto-Glo ECL (National Diagnostics), and
images were taken with either a Kodak-4000MM Image Station or iBright (Invitrogen).
2.7 Phylogenetic analysis
The dsRBDs protein sequences from 62 viral proteins were aligned using MUSCLE [49]. A
maximum likelihood phylogenetic tree was then generated from the multiple sequence alignment
using PhyML 3.0 with 100 bootstrap replicates [50]. The resulting tree was visualized using the
program FigTree v1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/).
2.8 Confocal microscopy
To determine the localization of PKR and CRV-157, HeLa-PKRkd cells were plated either on glass
coverslips in 12 well plates or on 35-mm coverglass bottom dishes (MatTek). At 40-50%
confluency, the cells were co-transfected with EGFP-tagged chicken PKR (0.5µg), and mCherry
tagged CRV157 (0.5µg) and incubated at 37˚C for 48h. After the incubation, the media was
removed, and cells were washed with PBS. The cells were then fixed with 2% formaldehyde and
incubated for 20-30 min, followed by PBS washing to remove formaldehyde. Following
formaldehyde removal, cells were incubated with 50mM glycine for 5-10 min at RT and then
washed with PBS. The cells were then incubated with nuclear stain, DAPI (300nM), for 15-20 min
and washed (x3) with PBS. Glass coverslips were removed and placed upside down on glass slides
with mounting media. Excess mounting media were absorbed from the edges using filter paper
and edges were sealed by applying nail polish. After nuclear staining, the cells on the glass bottom
dish were kept in PBS.
Samples were then observed with an inverted Carl Zeiss LSM 880 confocal microscope using
excitation wavelengths of 405nm (DAPI), 514nm (EGFP), and 633nm (mCherry) with 40x EC
Plan-Neofluar oil objective with a 1.3 numerical aperture (NA). Fluorescence was detected using
a gallium arsenide phosphide (GaAsP) detector to ensure improved signal to noise ratio.
Fluorescent images were obtained by sequential scanning of each channel to ensure reliable
quantitation of colocalization. Images of random fields were acquired, and channels were merged
in Zeiss Zen microscope software during acquisition, which allowed qualitative judgments about
colocalization of CRV157-mCherry and chicken PKR-EGFP signals. Confocal images were then
imported to Fiji (v2.00-rc-68/1.52f) [51] software for colocalization analysis.
2.9 Quantitative colocalization analysis (QCA)
Images acquired from the confocal microscope were processed using Fiji and the JACoP plugin
[51,52]. Briefly, the background of channels with mCherry and EGFP were subtracted from the
control confocal images (no fluorescent/fluorescence) by using the image calculator tool.
Furthermore, background was corrected using Coste’s automatic threshold for all channels.
Pearson’s correlation coefficients (PCC) were calculated using Pearson’s coefficient tab of the
JACoP plugin. PCC values range from -1.0 to 1.0, where -1.0 implies no overlap and 1.0 indicates
complete colocalization [53]. The data obtained were analyzed in Excel.
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3. Results
3.1 CRV157 contains a dsRNA-binding domain
In order to identify putative PKR inhibitors in other poxviruses, we performed PSI-BLAST
searches of poxvirus sequences using default parameters with homologs of E3 and K3 from various
poxviruses. For example, when we used the dsRNA binding domain of VACV E3 or the racoonpox
virus E3 homolog as bait, CRV157, a putative protein encoded by CRV was identified in iteration
2 but stayed below the PSI-BLAST threshold of 0.005 through iteration 4, when no new sequences
were identified above the threshold. This indicated that CRV157 might be a dsRNA binding
protein, distantly related to E3 orthologs. CRV157 was previously identified as a putative dsRNA
binding protein when CRV proteins of unknown functions were searched against the NCBI
database using PSI-BLAST [43]. The authors concluded that CRV157 was not an ortholog of the
E3 family. To follow up, we extended our search for E3 homologs to include all viral proteins in
the database. While dsRBDs from dozens of non-poxviruses were detected after iterations 2 and
3, CRV157 was only detected after iteration 4 and its ortholog (ORF198) from saltwater
crocodilepox virus (SwCRV) only after iteration 5. To study the relationship among viral dsRBDs,
we performed a phylogenetic analysis from a multiple sequence alignment containing dsRBDs
from various viruses. In this analysis, CRV157 and SwCRV198 were found in a different clade
than E3 sequences from other poxviruses (Fig. 1). Even though the basal branches were not
supported by high bootstrap values (a notable problem for phylogenetic analyses of dsRNA-
binding proteins because only a few amino acids are conserved [54]) these analyses, together with
the PSI-BLAST searches further support the notion that CRV157 does not share an immediate
ancestor with the poxvirus E3 family.
When PSI-BLAST searches with the full CRV157 sequences of the whole NR database were
performed with initial BLAST thresholds set to 1, various dsRBDs of ribonuclease III proteins
from Streptomyces species were discovered during the second iteration. The detected homology
was not restricted to the dsRBD but extended a further 45 amino acids into the C-terminus (Fig.
2A). A multiple sequence alignment with other dsRBDs including VACV E3 homologs myxoma
virus 029 and sheeppox virus 34, showed a deletion between beta sheets 1 and 2 in CRV157, as
previously reported [43]. Sequence identities calculated from the alignment including only the
dsRBDs showed highest sequence identity of CRV157 and SwCRV198 with S. gossypiisoli
ribonuclease III (Fig. 2B). Combined, these data indicate that CRV157 is not an ortholog of the
poxvirus E3 family, but originated independently.
3.2 CRV157 is a dsRNA binding protein.
Because CRV157 contains a deletion between beta sheets 1 and 2, also called region 2, which has
been shown to be important for dsRNA binding in other dsRBDs, we experimentally tested its
dsRNA-binding capability in a pull-down assay using the synthetic dsRNA analog
polyinosinic:polycytidylic acid (poly I:C). We assessed poly I:C pull-down of CRV157 and a
mutant of CRV157 (CRV157-K48A/K49A) in which key lysine residues that were shown to be
essential for dsRNA binding in other dsRBD have been replaced by alanine residues [40]{Bycroft,
1995 #51}[48,55,56]. In order to be able to detect and to pull-down the proteins, we added C-
terminal FLAG epitope tags to CRV157, CRV157-K48A/K49A, and E3L in the mammalian
expression vector pSG5. For the pull-down assay, HeLa-PKRkdcells were transiently transfected
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with either pSG5 vector, VACV E3L, CRV157, or CRV157-K48A/K49A. Cell lysates from these
cells were incubated with poly I:C-conjugated sepharose beads and bound proteins were subjected
to Western blot analysis. While all three proteins were detected in the initial lysates, only E3 and
CRV157 were detected in the pull-downs, but not CRV157-K48A/K49A (Fig. 3). These results
demonstrate that CRV157 is a dsRNA binding protein.
3.3 CRV157 is a PKR inhibitor
To test whether CRV157 can inhibit PKR, we used an established luciferase-based reporter (LBR)
assay [45]. In this assay, PKR, potential PKR inhibitors and a luciferase reporter are co-transfected
into PKR-depleted or PKR-deficient cells and luciferase activity is used as a proxy for translation,
where low luciferase activities indicate high PKR activity, whereas high luciferase activities
indicate low PKR activity. In this assay PKR is activated during transient transfection, likely by
dsRNA formed from overlapping RNA transcripts of the transfected plasmids [57]. We co-
transfected either empty vector, mouse or chicken PKR with empty vector, E3L, CRV157 or
CRV157-K48A/K49A. Co-transfection of both E3L and CRV157 resulted in increased luciferase
activities, which is indicative of PKR inhibition, whereas CRV157-K48A/K49A had no effect
(Fig. 4A). To analyze the contribution of the two CRV157 domains to PKR inhibition, we
generated FLAG-tagged constructs encoding either the N-terminal (dsRBD) or C-terminal
domains (Fig. 4B) and performed LBR assays with these and full-length constructs. The construct
containing the dsRBD resulted in comparable luciferase activity as full-length CRV157, whereas
the C-terminal domain did not affect PKR activity (Fig. 4C). Western blots of transfected cells
showed comparable expression of CRV157 and the N-terminal domain, whereas no expression
was observed for the C-terminal domain (Fig. 4D). Co-transfection of increasing amounts of
CRV157 and the CRV157 N-terminus with chicken PKR revealed that full-length CRV157
inhibited PKR more efficiently than the dsRBD alone (Fig. 4E).
3.4 CRV157 can inhibit PKR from multiple species
Multiple studies have previously shown that PKR inhibitors from poxviruses can show species
specific PKR inhibition [45,46,58-60]. To investigate whether CRV157 exhibits species-specific
PKR inhibition, we performed the LBR assay with CRV157 or VACV E3L as control, and PKR
from the following seven vertebrate species: human, mouse, European rabbit, pig, Armenian
hamster, Chinese hamster or chicken (Fig. 4.5). The PKR gene from the Nile crocodile, the natural
CRV host, has not yet been sequenced and was therefore unavailable for these experiments. All
tested PKRs were inhibited by E3 and CRV157, although to different extents. Human and mouse
PKR were only weakly to moderately inhibited, whereas chicken PKR was most strongly inhibited.
In all cases, E3 showed stronger inhibition than CRV157.
3.5 Colocalization of CRV157 with PKR
To investigate whether CRV157 colocalizes with PKR, we fluorescently labelled CRV157 and
chicken PKR at the C-termini, with mCherry (CRV157-mCherry) and EGFP (chicken PKR-
EGFP), respectively. We co-transfected HeLa-PKRkd cells with CRV157-mCherry and chicken
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PKR-EGFP to analyze their localization in cells using confocal microscopy. Confocal images were
taken for quantitative colocalization analysis (QCA), which measures the degree of spatial
coincidence between CRV157-mCherry and chicken PKR-EGFP using Fiji ImageJ analysis
software. The degree of co-localization of CRV157 and chicken PKR was calculated from at least
15 representative confocal images from three independent experiments and is shown as the average
of the Pearson’s correlation coefficients (PCC). The PCC value (0.77) from QCA indicates a strong
correlation for the colocalization of CRV157 and chicken PKR in the cell cytoplasm (Fig. 6A) [61].
Representative images of the localizations are shown (Fig. 6B).
3.6 CRV157 can rescue the replication of an attenuated VACV strain that lacks its PKR
inhibitors
The best test to analyze if CRV157 is a biologically meaningful PKR inhibitor, is to insert it into
a VACV strain that lacks its PKR inhibitors and test whether it can rescue replication. We therefore
inserted either CRV157, CRV157-K48A/K49A, or VACV E3L (as positive control) into the
replication incompetent VACV strain VC-R4, which lacks both E3L and K3L. All the transgenes
were integrated into VC-R4 by a method that allows seamless integration of transgenes into the
E3L locus and is driven by the same endogenous E3L promoter [47]. To compare the replication
efficiency of these viruses relative to VC-2 (parental to all viruses tested here), vP872 (∆K3L),
and the parental VC-R4 (∆E3L∆K3L), we infected rabbit kidney RK13 cells at MOI= 0.01 with
these viruses over the course of 96 hours (Fig. 7A). Cell lysates were collected at different time
points and viruses were titered on the permissive RK13+E3+K3 cell line. The replication kinetics
indicate that VC-R4 and VC-R4+CRV157-KK-AA were replication defective. VC-R4 containing
E3L (VC-R4+E3L) replicated comparably well as VC-2 and vP872 at all time points, which
reached plateaus after 48 hours. VC-R4 containing CRV157 (VC-R4+CRV157) showed
replication, but it lagged behind the replication of the E3L-containing viruses at 12, 24 and 48
hours by about 10-fold. However, replication was comparable at 72 and 96 hours to the E3L-
containing viruses. VC-R4+CRV157 replicated to more than 4 orders of magnitude higher titers
than VC-R4 and VC-R4:CRV157-KK-AA viruses after 72 and 96 hours.
Since eIF2a phosphorylation more directly demonstrates the effects of PKR inhibitors, we also
monitored eIF2a phosphorylation levels in virus-infected RK13 cells by Western blot analysis
(Fig. 7B). To analyze eIF2a phosphorylation, we infected RK13 cells with the above mentioned
VACV strains at MOI=3 for 6 hours. In RK13 cells, VC-R4+CRV157-K48A/K49A infection
induced high levels of eIF2a phosphorylation, which was comparable to that that seen in VC-R4
infected cells. Whereas, VC-2, vP872 and VC-R4+E3L infections led to low eIF2a
phosphorylation levels, intermediate eIF2a phosphorylation levels were observed in VC-
R4+CRV157 infected cells. Thus, PKR sensitivity and virus replication correlated well with eIF2a
phosphorylation levels in infected RK13 cells.
4. Discussion
Poxviruses infecting crocodilians pose threats for both farm-raised and wild populations [12,14,16].
To date, the full genome sequences from two such poxviruses (CRV and SwCRV) have been
described, which form a monophyletic clade and have been assigned to the crocodylidpoxvirus
10Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 May 2022 doi:10.20944/preprints202205.0393.v1
genus [10,11]. In depth genomic characterization of multiple SwCRV isolates, showed strong
evidence of recombination within the SwCRV clade [62]. To the best of our knowledge, however,
no functional characterizations of any crocodylidpoxvirus proteins have been reported. Here we
show that CRV157 is a PKR inhibitor and highlight the importance of the role of the PKR pathway
in crocodylidpoxvirus infections.
Together with an earlier study [43], all evidence presented here indicates that CRV157 evolved
independently from the other poxvirus PKR inhibitors. While it is hard to trace the evolutionary
origins of individual dsRBDs because of the rapid evolution of the dsRBD having only a few
conserved amino acid residues [54], the extended homology in the C-terminus of CRV157 with
Streptomyces sp. RNase III indicates that it might have been horizontally transferred from an
unsampled bacterium. However, the currently available sequences do not allow us to infer the
origin of CRV157 with high confidence. While most poxvirus proteins show higher sequence
identities with eukaryotic proteins in bioinformatic analyses, some poxvirus proteins appear to be
more similar to bacterial proteins indicating possible transfer events between bacteria and viruses
[63]. Indeed, several CRV proteins show higher sequence identities to bacterial proteins than to
eukaryotic proteins [10]. One of these examples is CRV051, which shows highest sequnce
identities (about 32%) with prokaryotic serine recombinases [64].
Although CRV157 evidently contains a dsRBD as previously reported [43], it was important to
determine, if it was actually functional, especially in light of a deletion between beta sheets 1 and
2 (region 2), which includes amino acid residues that have been shown to be involved in dsRNA
interactions in other dsRBDs [65]. Poly (I:C) pull-down of wild type CRV157, but not of CRV157-
K48A/K49A, showed that CRV157 is a dsRNA binding protein. This binding activity correlated
with the inhibition of PKR, which was only inhibited by wild type CRV157 but not CRV157-
K48A/K49A. Importantly, CRV157, but not CRV157-K48A/K49A, was able to rescue replication
of VACV missing K3L and E3L genes in RK13 cells. The finding that titers of the CRV157-
containing virus were about 10-fold lower out to 3 days of infection than of the E3L-containing
viruses, however, indicated that CRV157 inhibited rabbit PKR sub-optimally, at least in the
context of VACV infection. This correlated with the intermediate eIF2a phosphorylation levels
induced by VC-R4+CRV157, and the findings that CRV157 was not as potent in inhibiting PKR
from all tested vertebrates as VACV E3 in the LBR assay. It is possible that PKR from the natural
host, the Nile crocodile, might be more sensitive to CRV157 inhibition, which could not be tested
because the PKR gene from this species is not known, or that it works sub-optimally in the context
of VACV infection, or in the tested mammalian cells. It is also possible that the intermediate PKR
inhibition might be associated with the longevity of the CRV infection in Nile crocodiles, which
can often persist for many months [6,12]. Another possibility is that intermediate PKR inhibition
may have unexpected consequences such as the functional activation of the pro-inflammatory NF-
kB pathway, as recently shown [36]. In this case, limited inhibition of translation resulted in NF-
kB activation and the translation of induced mRNA, whereas no PKR inhibition abolished
translation of the induced mRNAs.
An interesting observation from the LBR assay was that the individual tested PKRs appeared to
exhibit differential sensitivity to both CRV157 and E3, with human PKR being inhibited least and
chicken PKR being inhibited strongest, indicating that some PKRs might be more sensitive to viral
dsRNA binding proteins than others. Notably, expression of the dsRBD of CRV157 alone
(CRV157 del C) did not inhibit PKR as efficiently as full length CRV157, indicating that the
uncharacterized C-terminal domain of CRV157 might be also involved in PKR inhibition. This is
reminiscent of the situation found in VACV E3 and the variola virus E3 homolog, for which the
11Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 May 2022 doi:10.20944/preprints202205.0393.v1
N-terminal Za domain was shown to be important for PKR inhibition in addition to the C-terminal
dsRBD in yeast assays [41,66]. In the context of vaccinia virus infection, an N-terminally truncated
E3L gene resulted in altered eIF2a phosphorylation levels at 6 hours, but led to increased eIF2a
phosphorylation at 9 and 12 hours after infection [67]. These findings show that PKR inhibition
can be influenced by domains adjacent to the dsRBD.
5. Conclusions
Poxviruses evolved multiple inhibitors of antiviral host proteins, often targeting various steps in
antiviral pathways [19]. The critical role of PKR in inhibiting virus replication is highlighted by
the fact that most mammalian poxviruses contain two inhibitors, named E3 and K3 in vaccinia
virus, which either prevent PKR activation, or PKR interaction with its substrate IF2a. Notably,
E3 and K3 orthologs are missing in avipoxviruses and crocodylidpoxviruses [37,38].
Avipoxviruses, possess a protein with homology to the eIF2a-binding and protein phosphatase 1
(PP1)-binding region of the DNA damage-inducible protein 34 (GADD34). Similar to GADD34,
canarypox virus protein 231, was able to suppress PKR-toxicity in a yeast assay, in which it also
promoted PKR-mediated eIF2a dephosphorylation [68]. The data presented here show that
CRV157 is a fourth PKR inhibitor found in poxviruses and thus provides a compelling example
for convergent evolution.
Because of the negative impact of crocodilepox viruses on crocodile farming, an important goal
would be to develop an animal vaccine for this industry. Because vaccinia virus with targeted
deletion in the PKR inhibitor E3L has been shown to protect mice and rabbits from lethal
challenges with ectromelia virus and rabbitpox virus, respectively [69,70], CRV or SwCRV strains
with deletions in CRV157 or SwCRV198 might be promising candidates for such vaccines.
Acknowledgments:
Author Contributions: Conceptualization, M.J.R. and S.R.; methodology, M.J.R., L.T., S.H. and
S.R.; validation, M.J.R.; formal analysis, M.J.R., L.T., and S.R.; investigation, M.J.R., L.T., S.H.
and S.R.; resources, M.J.R., L.T., S.H. and S.R. and T.S.; data curation, M.J.R., L.T., and S.R.;
writing— original draft preparation, M.J.R. and S.R.; writing and editing, M.J.R., L.T., S.H. and
S.R.; visualization, M.J.R., L.T., and S.R.; supervision, S.R.; project administration, S.R.; funding
acquisition, S.R. All authors have read and agreed to the published version of the manuscript.
Funding: This research was partially funded by grant AI114851 (to S.R.) from the National
Institute of Allergy and Infectious Diseases, National Institutes of Health, and start-up funds from
the University of California, Davis (to S.R.).
Conflicts of Interest: All authors declare no conflict of interest.
12Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 May 2022 doi:10.20944/preprints202205.0393.v1
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17Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 May 2022 doi:10.20944/preprints202205.0393.v1
Figures
Rotavirus
045 J NSP1 YP_010086031.1
Bat
042 rotavirus NSP1 QRV11667.1
90 Porcine
041 rotavirus H NGS-14 NSP1 BBC18340.1
Rotavirus
040 H SP-VC18 NSP1 QKY66963.1
Bat
0 4 3 rotavirus NSP1 QTE76090.1
Bat
0 4 4 rotavirus H NSP1 AWV67033.1
Rotavirus
038 I KE528/2012 NSP1-2 AKA63280.1
Rotavirus
039 I SD-F3 NSP1 ULF48556.1
100 Nile
001 crocodilepox virus 157 YP_784347.1
Saltwater
002 crocodilepox virus 198 QGT47498
97 Human
019 rotavirus C NSP3 AGO58054.2
Human
020 rotavirus C NSP3 AAX21224.1
76 Bovine
022 rotavirus C NSP3 BCU08699.1
74 Rotavirus
017 C NSP3 AXQ59463.1
67 Porcine
021 rotavirus C NSP3 BCU08769.1
Porcine
018 rotavirus B NSP2 QBJ02236.1
Beihai
027 shrimp virus 2 gp2 YP_009333523.1
79 Drosophila
026 C virus ORF1 AWI97853.1
Wenzhou
025 picorna-like virus 47 APG78496.1
Mimivirus
032 LCMiAC02 3'-5' exonuclease QBK89441.1
Kadipiro
024 virus VP8 APG79137.1
92 Satyrvirus
035 sp. ribonuclease 3 AYV85794.1
50
71 Moumouvirus
030 maliensis RNAse QGR54010.1
Megavirus
031 courdo7 ribonuclease 3 AEX61722.1
74
Dishui
047 Lake phycodnavirus 3 ribonuclease III QIG59698.1
Dishui
037 Lake phycodnavirus 4 RNAse III QIG59958.1
53 Micromonas
046 pusilla virus PL1 ribonuclease III AET43609.1
87 Micromonas
036 pusilla virus SP1 RNAse III YP_009664928.1
Micromonas
049 pusilla virus PL1 ribonuclease III AET43609.1
Ostreococcus
048 tauri virus RT-2011 OtV6_130 AFC35038.1
Ostreococcus
028 lucimarinus virus 1 OlV1_149c YP_004061782.1
Ostreococcus
029 tauri virus 1 OTV1_125 YP_003212948.1
Bufonid
016 herpesvirus 1 ORF25 YP_009552888.1
54 Armadillidium
003 vulgare iridescent virus Ribonuclease III YP_009046792.1
100 Iridovirus
004 Liz-CrIV 340R QEA08313.1
Invertebrate
006 iridescent virus 6 340R NP_149803.1
85 Invertebrate
007 iridovirus 25 Ribonuclease III YP_009010659.1
Anopheles
005 minimus iridovirus Ribonuclease III YP_009021208.1
011 Hubei picorna-like virus 55 AWS06670.1
87 012
Changjiang crawfish virus 6 YP_009336630.1
75 Changjiang
034 crawfish virus 6 APG78044.1
Hubei
008 picorna-like virus 56 YP_009336543.1
Sitobion miscanthi virus 1 polyprotein QCI31816.1
010
68 80
Rosy
013 apple aphid virus polyprotein ABB89048.1
57 Acyrthosiphon
009 pisum virus protein P1 QAA78869.1
63 Acyrthosiphon
033 pisum virus P1 QAA78871.1
57 Solenopsis
015 invicta virus 7 polyprotein QBL75890.1
Averyanov
023 virus RNA-dependent RNA polymerase QIS87995.1
Beihai
014 anemone virus 1 BVE94_gp2 YP_009333203.1
Pseudocowpox virus 020 AFA41730.1
90 0 6 0
50 Orf
053 virus OV20.0 CAA10953.1
Seal
058 parapoxvirus 013 YP_009389309.1
51 Swinepox
054 virus 032 NP_570192.1
Myxoma
052 virus M156 NP_051743.1
54 Tanapox
051 virus 34 ABQ43508.1
53 52 Deerpox
061 virus W-1170-84 042 ABI99027.1
Goatpox
055 virus Pellor 030 YP_001293225.1
96 Cotia
062 virus SPAn232 051 YP_005296239.1
50 BeAn
056 58058 virus E3 YP_009329659.1
73 Yokapox
057 virus 037 YP_004821390.1
83 Vaccinia
050 virus Copenhagen E3 P21081.1
Raccoonpox
059 virus 054 YP_009143366.1
0.6
Figure 1. Phylogenetic relationship between viral dsRNA-binding domains.
A maximum phylogenetic tree of dsRBDs from indicated viral proteins was constructed from a
multiple sequence alignment generated with MUSCLE using PhyML. Numbers next to branches
indicate bootstrap values ≥ 50. Accession numbers of proteins are indicated.
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